The broad long-term objectives of this proposal are to study the role of iron in bacterial pathogenesis, specifically focusing on the pathogen, Vibrio vulnificus. Iron plays a very important role in the pathogenesis of V. vulnificus infections. Patients with iron overload syndromes, such as alcoholic liver disease, cirrhosis and hemochromatosis are particularly susceptible to infections with V. vulnificus . Epidemiological studies suggest that V. vulnificus accounts for over 50% of all vibrio-associated illness in this country. V. vulnificus septicemia has a 50% mortality rate and causes about 15 deaths per year, one of the major causes of death due to foodborne illness. Studying the role of iron in V. vulnificus infections should help define the importance of iron in bacterial virulence. The specific aims of the project are: 1.) To clone and characterize the genes encoding the two major iron-regulated outer membrane proteins of V. vulnificus, the vulnibactin receptor, and the heme receptor. 2.) To examine the roles of heme utilization and catechol siderophore synthesis and uptake in V. vulnificus pathogenesis; and 3.) To examine the effect of iron overload on the ability of macrophages to phagocytize and kill V. vulnificus. The experimental design includes cloning the vulnibactin and heme receptor for V. vulnificus. These genes will be analyzed for regulation by iron and the ferric uptake regulatory protein (Fur). V. vulnificus mutants will be constructed in which these genes are inactivated . The mutants will be examined for loss of virulence and the ability to acquire host iron sources. The effect of iron overload on phagocytosis and intracellular killing of V. vulnificus and its mutants will be assessed in iron-loaded and normal macrophages. The methods to be used include using mixtures of oligonucleotide probes derived from the N-terminal protein sequence of the outer membrane proteins (vulnibactin receptor and heme receptor) to screen a genomic DNA library in order to clone these genes. Mutants lacking the vulnibactin receptor and the heme receptor will be constructed by in vivo marker exchange. Utilization of host iron sources will be assessed by a plate assay and kinetic studies. LacZ fusions and Northern blots will be used to analyze the transcriptional regulation of these genes by iron and Fur. The virulence of the V. vulnificus iron acquisition and outer membrane mutants will be analyzed using an infant mouse model of infection and adult iron-loaded and normal mouse models. Phagocytosis and killing will be assessed using an acridine orange technique.